Methods and devices for promoting endothelial morphogenesis

ABSTRACT

In one aspect the present invention provides methods for promoting endothelial morphogenesis. The methods of this aspect of the invention include the step of providing to one or more endothelial cells an amount of an osteoprotegerin sufficient to promote endothelial morphogenesis. The methods of this aspect of the invention can be practiced in vivo or in vitro. In another aspect, the present invention provides implantable medical devices that each include: (a) a device body; and (b) a layer attached to a surface of the device body, the layer comprising a molecule selected from the group consisting of osteoprotegerin and a nucleic acid molecule encoding osteoprotegerin, wherein the device is adapted to be completely or partially implanted into an animal body. The implanted medical device thus promotes the growth of blood vessels in the surrounding tissue, thereby reducing or preventing the formation of a collagenous capsule around the implanted medical device.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 10/142,658,filed May 9, 2002, which claims the benefit of Application No.60/290,230, filed on May 10, 2001.

GOVERNMENT RIGHTS

This invention was made with government support under grant numberEEC-9529161 awarded by National Science Foundation; and grant numberHL18645, awarded by National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods for promoting the growth ofblood vessels.

BACKGROUND OF THE INVENTION

The treatment of many diseases requires the growth of new blood vesselseither in vivo or in vitro.

For example, during a heart attack the blood supply to a portion of theheart muscle is interrupted and the affected muscle dies or is injured.A goal of medical research is to develop methods to promote the growthof new blood vessels into, and around, the damaged heart tissue to aidits recovery.

Again by way of example, there is a continuing need for methods thatpromote the formation of blood vessels in vitro. These cultured bloodvessels can be used to surgically repair or replace damaged bloodvessels in an animal body. For example, cultured blood vessels can beused to repair blood vessel aneurisms.

Again by way of example, the implantation of a medical device into ananimal body elicits a wound response. This type of wound response iscalled the foreign body response and results in the encapsulation of theimplant by a poorly-vascularized, collagenous, capsule that cancompromise the function of the implant. Formation of the collagenouscapsule can be slowed or prevented by promoting the growth of bloodvessels in the tissue surrounding the implanted device. There is,therefore, a continuing need for methods that promote the growth ofblood vessels into the tissue surrounding implanted medical devices.

Large blood vessels, such as arteries and veins, include an endotheliallining surrounded by other tissue layers, such as a layer of musclecells that regulate the diameter of the vessel, and thereby regulateblood pressure within the vessel. Some small blood vessels, such ascapillaries, are made entirely from endothelial cells. The presentinvention provides methods for promoting endothelial morphogenesis,which is the formation of an animal anatomical structure fromendothelial cells. Thus, the methods of the present invention can beused to promote the formation of new blood vessels, such as capillaries,that are composed entirely of endothelial cells; and also can be used topromote the formation of endothelial portions of blood vessels, such aspromoting the formation of an endothelial lining in the lumen of anartificial blood vessel.

The present invention also provides implantable medical devices thatpromote endothelial morphogenesis, including the formation of new bloodvessels, in the living tissue that completely, or partially, surroundsthe device after implantation into an animal body. As described morefully herein, the methods and devices of the invention utilizeosteoprotegerin protein, or biologically active fragments thereof, topromote endothelial morphogenesis.

SUMMARY OF THE INVENTION

In accordance with the foregoing, in one aspect the present inventionprovides methods for promoting endothelial morphogenesis. The methods ofthis aspect of the invention include the step of providing to one ormore endothelial cells an amount of an osteoprotegerin sufficient topromote endothelial morphogenesis. In this context, the term“endothelial cells” encompasses endothelial stem cells anddifferentiated endothelial cells. Examples of endothelial morphogenesisinclude the formation of capillaries from endothelial cells, and theformation of the endothelial lining of arteries and veins. The methodsof this aspect of the invention can be practiced in vivo or in vitro.Exemplary methods for providing osteoprotegerin to one or moreendothelial cells include: direct injection of osteoprotegerin into, orclose to, a target site within an animal body; secretion ofosteoprotegerin from cells adjacent to the target endothelial cells(e.g., by grafting tissue that secretes osteoprotegerin onto, or closeto, the target endothelial cells); or by attachment of osteoprotegerinto a surface of a medical device that is implanted at, or close to, thesite of the target endothelial cells within an animal body.

As described more fully herein, the methods of the invention are usefulin any situation where promotion of endothelial morphogenesis isdesired, such as promotion of blood vessel growth in and around damagedheart muscle.

In another aspect, the present invention provides implantable medicaldevices that each include: (a) a device body; and (b) a layer attachedto a surface of the device body, the layer comprising a molecule(typically a multiplicity of molecules) selected from the groupconsisting of (1) a nucleic acid molecule encoding osteoprotegerin(e.g., the nucleic acid molecule having the sequence set forth in SEQ IDNO: 1), and (2) osteoprotegerin (e.g., the osteoprotegerin having thesequence set forth in SEQ ID NO:2), wherein the device is adapted to becompletely or partially implanted into an animal body. The implantedmedical device promotes the growth of blood vessels in the surroundingtissue, thereby reducing or preventing the formation of a collagenouscapsule around the implanted medical device. As described more fullyherein, the implantable medical devices of the invention are useful inany situation where promotion of endothelial morphogenesis is desired,such as promotion of blood vessel growth in tissue surrounding theimplanted device, thereby reducing or preventing the foreign bodyreaction.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows a perspective view of a representative medical device ofthe invention with a portion of the surface layer removed to expose theunderlying device body.

FIG. 2 shows a transverse cross-section of the medical device of FIG. 1.

FIG. 3 shows the porous matrix structure of the surface layer of therepresentative medical device shown in FIG. 1.

FIG. 4 shows the number of blood vessels (per visual field when viewedunder a microscope at 400× magnification) growing into polyvinyl alcoholsponges implanted into mice. The sponges had been soaked in one of thefollowing solutions: a solution of bovine fibroblast growth factor(bFGF); a solution of the Fc portion of human IgG (FC); and a solutionof a hybrid protein composed of the Fc portion of human IgG fused toosteoprotegerin (OPG-FC). The control was a sponge soaked in phosphatebuffered saline (PBS). This experiment is described in Example 1.

FIG. 5 shows the number of blood vessels growing from excised portionsof mouse aortic arch into collagen gel containing osteoprotegerin (OPG),and the number of blood vessels growing from excised portions of mouseaortic arch into control collagen gel that does not containosteoprotegerin. This experiment is described in Example 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless specifically defined herein, all terms used herein have the samemeaning as they would to one skilled in the art of the presentinvention. Practitioners are particularly directed to Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Press, Plainsview, N.Y. (1989), for definitions and terms of theart. Unless stated otherwise, all publications and patents that arecited in the present patent application are incorporated herein byreference in their entirety.

As used herein, the term “endothelial morphogenesis” means the formationof an animal anatomical structure (e.g., capillaries and the endotheliallining of the lumen of arteries and veins) from endothelial cells. Inthis context, the term “endothelial cells” encompasses endothelial stemcells and differentiated endothelial cells.

As used herein, the term “osteoprotegerin”, refers to an art-recognizedclass of proteins (and their functional sequence variants) that aremembers of the tumor necrosis factor receptor (TNFR) superfamily, andthat possess several biological activities in vivo, including blockingbone resorption, promoting bone formation, and promoting endothelialmorphogenesis. Osteoprotegerins are reviewed by Schoppet et al.,Arteriosclerosis, Thrombosis, Vascular Biology 22:549-553 (2002).

As used herein, the term “percent identity” or “percent identical”, whenused in connection with osteoprotegerin molecules useful in the practiceof the present invention, is defined as the percentage of amino acidresidues in an osteoprotegerin molecule sequence that are identical withthe amino acid sequence of a specified osteoprotegerin molecule (such asthe amino acid sequence of SEQ ID NO:2), after aligning theosteoprotegerin sequences to achieve the maximum percent identity. Whenmaking the comparison, no gaps are introduced into the osteoprotegerinsequences in order to achieve the best alignment.

Amino acid sequence identity can be determined, for example, in thefollowing manner. The amino acid sequence of an osteoprotegerin molecule(e.g., the amino acid sequence set forth in SEQ ID NO:2) is used tosearch a protein sequence database, such as the GenBank database(accessible at web site http://www.ncbi.nln.nih.gov/blast/), using theBLASTP program. The program is used in the ungapped mode. Defaultfiltering is used to remove sequence homologies due to regions of lowcomplexity. The default parameters of BLASTP are utilized.

As used herein, the term “implantable medical device” refers to medicaldevices that are adapted to be implanted into the body of an animal,such as a mammal, including a human, during the normal operation of thedevice. The devices may be completely or partially implanted into thebody of an animal.

In one aspect, the present invention provides methods for promotingendothelial morphogenesis. The methods each include the step ofproviding to one or more endothelial cells an amount of anosteoprotegerin protein sufficient to promote endothelial morphogenesis.In this context, the term “endothelial cells” encompasses differentiatedendothelial cells and endothelial stem cells. The methods of this aspectof the invention can be used in any situation in which promotion ofendothelial morphogenesis is desired either in vivo or in vitro. Thefollowing are representative examples of useful applications of themethods of this aspect of the invention. The methods of this aspect ofthe invention can be used to promote formation of blood vessels, such ascapillaries, in vivo, such as in and around an area of mammalian heartmuscle that has been damaged, such as by reduced blood flow resultingfrom a heart attack. The methods of this aspect of the invention can beused to promote formation, in vivo, of the endothelial lining of thelumen of veins or arteries; and can be used, in vitro, to promote theformation of an endothelial lining in the lumen of an artificial bloodvessel, such as a hollow tube made from a biocompatible material such asDacron®. In this regard, the following publications disclose methodsthat can be used to make artificial blood vessels, including artificialblood vessels that include one or more layers of endothelial cells:Griffith, L. G. and Naughton, G., Science 295(5557):1009-1014 (2002);and, L'Heureux, et al., Science 284(5420):1621-1622 (1999). Again by wayof example, the methods of this aspect of the invention can be used topromote the formation of blood vessels, such as capillaries, in thetissue surrounding an implanted medical device.

Any osteoprotegerin protein that promotes endothelial morphogenesis isuseful in the methods of the present invention. Osteoprotegerin proteinsuseful in the methods of the present invention include naturallypurified osteoprotegerin protein, chemically synthesized osteoprotegerinprotein, and osteoprotegerin protein produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, insect, mammalian, avian and higher plant cells.Osteoprotegerin fragments that promote endothelial morphogenesis arealso useful in the practice of the present invention. For example,osteoprotegerin fragments that include the first four structural domains(located within the first 200 amino acids, counted from the N-terminus,of the complete osteoprotegerin amino acid sequence), as identified byK. Yamaguchi et al., J. Biol. Chem. 273(9):5117-5123 (1998), are usefulin the practice of the present invention.

Osteoprotegerin, or osteoprotegerin fragments, can be recovered andpurified by any useful purification method, including ammonium sulfateor ethanol precipitation, acid extraction, anion or cation exchangechromatography, gel filtration, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, and highperformance liquid chromatography. For example, a cDNA molecule encodingan osteoprotegerin can be expressed in microbial cells, such as E. colicells, and purified therefrom. Art-recognized techniques for thepurification of proteins and peptides are set forth, for example, inMethods in Enzymology, Vol. 182, Guide to Protein Purification, MurrayP. Deutscher, ed (1990).

The cDNA molecule set forth in SEQ ID NO:1 encodes a representativeosteoprotegerin (consisting of the amino acid sequence set forth in SEQID NO:2) useful in the practice of the invention. Other representativeexamples of useful osteoprotegerin proteins include osteoprotegerinproteins that are at least 70% identical (such as at least 80%identical, or such as at least 90% identical, or such as at least 95%identical) to the osteoprotegerin protein consisting of the amino acidsequence set forth in SEQ ID NO:2.

In the practice of the invention, osteoprotegerin, or osteoprotegerinfragments, can be provided to endothelial cells in vivo by any usefulmeans, such as by one or more of the following, representative, methods.Osteoprotegerin, or osteoprotegerin fragments, can be introduced intothe body by injection at, or close to, the target endothelial cells.Nucleic acid molecules (e.g., cDNA molecules) encoding osteoprotegerincan be delivered into the body of an animal where they are taken up byendothelial cells and expressed therein, or are taken up by cellsadjacent to endothelial cells and expressed therein, and the expressedosteoprotegerin secreted so that it stimulates endothelial morphogenesisof the adjacent endothelial cells. The nucleic acid molecules encodingosteoprotegerin can be introduced into the body in the bloodstream, orby injection at, or close to, the target endothelial cells. Geneticallyengineered cells that secrete osteoprotegerin can be introduced close tothe target endothelial cells, for example by grafting geneticallyengineered tissue that secretes osteoprotegerin close to the targetendothelial cells. Osteoprotegerin protein (or osteoprotegerin fragmentsthat retain the ability to promote endothelial morphogenesis) can beintroduced to a desired location within an animal body by implantinginto the body of an animal a structure comprising osteoprotegerin, orosteoprotegerin peptides retaining the ability to promote endothelialmorphogenesis, disposed on a surface of the structure that contactstissue of the animal body when the structure is implanted therein.Similarly, nucleic acid molecules that encode osteoprotegerin, orosteoprotegerin peptides retaining the ability to promote endothelialmorphogenesis, can be non-covalently attached to the surface of animplantable structure which, after implantation into the body, releasesthe nucleic acid molecules, which are then taken up by adjacentendothelial cells wherein the encoded osteoprotegerin is expressed. Forexample, representative, art-recognized, methods for introducingosteoprotegerin protein, or osteoprotegerin fragments retaining theability to promote endothelial morphogenesis, or nucleic acid moleculesencoding osteoprotegerin or fragments thereof, into heart tissue arereviewed by M. J. Post et al., Cardiovascular Research 49:522-531(2001).

Osteoprotegerin protein, or osteoprotegerin peptides retaining theability to promote endothelial morphogenesis, can be delivered into thebody of an animal by any suitable means. By way of representativeexample, osteoprotegerin protein, or fragments thereof, can beintroduced into an animal body by application to a bodily membranecapable of absorbing the protein, for example the nasal,gastrointestinal and rectal membranes. The protein is typically appliedto the absorptive membrane in conjunction with a permeation enhancer.(See, e.g., V. H. L. Lee, Crit. Rev. Ther. Drug Carrier Syst. 5:69(1988); V. H. L. Lee, J. Controlled Release 13:213 (1990); V. H. L. Lee,Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991);A. G. DeBoer et al., J. Controlled Release 13:241 (1990)). For example,STDHF is a synthetic derivative of fusidic acid, a steroidal surfactantthat is similar in structure to the bile salts, and has been used as apermeation enhancer for nasal delivery. (W. A. Lee, Biopharm. Nov./Dec.,22, 1990).

The osteoprotegerin protein, or fragments thereof, may be introduced inassociation with another molecule, such as a lipid, to protect theprotein from enzymatic degradation. For example, the covalent attachmentof polymers, especially polyethylene glycol (PEG), has been used toprotect certain proteins from enzymatic hydrolysis in the body and thusprolong half-life (F. Fuertges, et al., J. Controlled Release, 11:139(1990)). Many polymer systems have been reported for protein delivery(Y. H. Bae, et al., J. Controlled Release 9:271 (1989); R. Hori, et al.,Pharm. Res. 6:813 (1989); I. Yamakawa, et al., J. Pharm. Sci. 79:505(1990); I. Yoshihiro, et al., J. Controlled Release 10:195 (1989); M.Asano, et al., J. Controlled Release 9:111 (1989); J. Rosenblatt et al.,J. Controlled Release 9:195 (1989); K. Makino, J. Controlled Release12:235 (1990); Y. Takakura et al., J. Pharm. Sci. 78:117 (1989); Y.Takakura et al., J. Pharm. Sci. 78:219 (1989)).

For transdermal applications, the osteoprotegerin protein, or fragmentsthereof, may be combined with other suitable ingredients, such ascarriers and/or adjuvants. There are no limitations on the nature ofsuch other ingredients, except that they must be pharmaceuticallyacceptable and efficacious for their intended administration, and cannotdegrade the activity of the active ingredients of the composition.Examples of suitable vehicles include ointments, creams, gels, orsuspensions, with or without purified collagen. The osteoprotegerinprotein, or fragments thereof, also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

Again by way of example, in the practice of the invention,osteoprotegerin, or osteoprotegerin fragments, can be provided toendothelial cells by delivery of nucleic acid molecules encodingosteoprotegerin, or a biologically active fragment thereof, which aretaken up by endothelial cells (or cells adjacent to the targetendothelial cells), and expressed therein. If the nucleic acid moleculesare taken up and expressed by cells adjacent to the target endothelialcells, then the expressed osteoprotegerin is secreted and interacts withthe target endothelial cells to promote endothelial morphogenesis. Thenucleic acid molecule can also be introduced into host cells, in vitro,and the modified cells introduced into the body of an animal (e.g., bygrafting) wherein they express and secrete osteoprotegerin.

Examples of nucleic acid molecules that encode osteoprotegerin, orfragment thereof, and that are useful in the methods of the invention(and in the devices of the invention) include nucleic acid moleculesthat encode an osteoprotegerin, or fragment thereof, and that hybridizeunder conditions of 5×SSC at 60° C. for 30 minutes to the complement ofthe nucleic acid molecule set forth in SEQ ID NO:1. Some nucleic acidmolecules that encode an osteoprotegerin and that are useful in thepractice of the present invention hybridize under conditions of 2×SSC at60° C. for 30 minutes to the complement of the nucleic acid molecule setforth in SEQ ID NO:1. Some nucleic acid molecules that encode anosteoprotegerin and that are useful in the practice of the presentinvention hybridize under conditions of 1×SSC at 60° C. for 30 minutesto the complement of the nucleic acid molecule set forth in SEQ ID NO:1.Some nucleic acid molecules that encode an osteoprotegerin and that areuseful in the practice of the present invention hybridize underconditions of 0.5×SSC at 60° C. for 30 minutes to the complement of thenucleic acid molecule set forth in SEQ ID NO:1.

Hybridization can be conducted, for example, by utilizing the techniqueof hybridizing radiolabelled nucleic acid probes to nucleic acidsimmobilized on nitrocellulose filters or nylon membranes as set forth atpages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2ndedition), J. Sambrook, E. F. Fritsch and T. Maniatis eds, the citedpages of which are incorporated herein by reference. An exemplaryhybridization protocol is set forth in Example 3 herein.

The nucleic acid molecule is typically part of a vector that typicallyincludes all of the necessary elements for expression of the encodedosteoprotegerin, or encoded osteoprotegerin fragment. Any art-recognizednucleic acid delivery method can be used to introduce a vector into oneor more cells for expression therein, including: transduction,transfection, transformation, direct injection, electroporation,virus-mediated gene delivery, amino acid-mediated gene delivery,biolistic gene delivery, lipofection and heat shock. See, generally,Sambrook et al, supra. Representative, non-viral, methods of genedelivery into cells are disclosed in Huang, L., Hung, M-C, and Wagner,E., Non-Viral Vectors for Gene Therapy, Academic Press, San Diego,Calif. (1999).

Expression vectors useful for expressing osteoprotegerin protein, orbiologically active fragments thereof, include chromosomal, episomal,and virus-derived vectors, e.g., vectors derived from bacterialplasmids, bacteriophages, yeast episomes, yeast chromosomal elements,viruses such as baculoviruses, papova viruses, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as cosmids andphagemids. In certain embodiments in this regard, the vectors providefor specific expression, which may be inducible and/or celltype-specific. Among such expression vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

For example, a coding sequence for osteoprotegerin, or a biologicallyactive fragment thereof, can be introduced into cells in situ, or afterremoval of the cells from the body, by means of viral vectors. Forexample, retroviruses are RNA viruses that have the ability to inserttheir genes into host cell chromosomes after infection. Retroviralvectors have been developed that lack the genes encoding viral proteins,but retain the ability to infect cells and insert their genes into thechromosomes of the target cell (see, A. D. Miller, Hum. Gen. Ther.1:5-14 (1990)). Adenoviral vectors are designed to be administereddirectly to patients. Unlike retroviral vectors, adenoviral vectors donot integrate into the chromosome of the host cell. Instead, genesintroduced into cells using adenoviral vectors are maintained in thenucleus as an extrachromosomal element (episome) that persists for alimited time period. Adenoviral vectors will infect dividing andnon-dividing cells in many different tissues in vivo including airwayepithelial cells, endothelial cells, hepatocytes and various tumors (B.C. Trapnell, Adv Drug Del Rev. 12:185-199 (1993)).

Another useful viral vector is the herpes simplex virus; a large,double-stranded DNA virus. Recombinant forms of the vaccinia virus canaccommodate large inserts and are generated by homologous recombination.To date, this vector has been used to deliver, for example, interleukins(ILs), such as human IL-1β and the costimulatory molecules B7-1 and B7-2(G. R. Peplinski et al., Ann. Surg. Oncol. 2:151-9 (1995); J. W. Hodgeet al., Cancer Res. 54:5552-55 (1994)).

A plasmid vector can be introduced into mammalian cells by anyart-recognized means, such as in a precipitate, such as a calciumphosphate precipitate, or in a complex with a charged lipid (e.g.,LIPOFECTAMINE™; Life Technologies, Inc.; Rockville, Md.) or in a complexwith a virus (such as an adenovirus) or components of a virus (such asviral capsid peptides). If the vector is a virus, it may be packaged invitro using an appropriate packaging cell line and then transduced intohost cells.

For example, a vector may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, or a gene activated collagenmatrix. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self-rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers.

Some liposomes have improved serum stability and circulation half-times(see, e.g., U.S. Pat. No. 5,741,516). Furthermore, various methods ofliposome and liposome-like preparations as potential drug carriers havebeen reviewed (see, e.g., U.S. Pat. Nos. 5,567,434; 5,552,157;5,565,213; 5,738,868 and 5,795,587).

Additionally, studies have demonstrated that intramuscular injection ofplasmid DNA formulated with 5% PVP (50,000 kDa) increases the level ofreporter gene expression in muscle as much as 200-fold over the levelsfound with injection of DNA in saline alone (R. J. Mumper et al., Pharm.Res. 13:701-709 (1996); R. J. Mumper et al., Proc. Intern. Symp. Cont.Rol. Bioac. Mater. 22:325-326 (1995)). Intramuscular administration ofplasmid DNA results in gene expression that lasts for many months (J. A.Wolff et al., Hum. Mol. Genet. 1:363-369 (1992); M. Manthorpe et al.,Hum. Gene Ther. 4:419-431 (1993); G. Ascadi et al., New Biol. 3:71-81(1991), D. Gal et al., Lab. Invest. 68:18-25 (1993)).

Various devices have been developed for providing nucleic acid molecules(e.g., DNA) to a target cell. One approach is to contact the target cellphysically with a catheter that directs nucleic acid molecules to thetarget cell; or to contact the target cell with an implantable materialthat includes nucleic acid molecules disposed therein or thereon (see,G. D. Chapman et al., Circulation Res. 71:27-33 (1992)). Anotherexemplary method for providing nucleic acid molecules to a target cellinvolves using a fibrous collagen implant material that is soaked in asolution of nucleic acid molecules (e.g., DNA) before being placed atthe target site.

Another approach is to utilize needle-free, jet injection devices whichproject a column of liquid directly into the target tissue under highpressure. (P. A. Furth et al., Anal. Biochem. 20:365-368 (1992); H. L.Vahlsing et al., J. Immunol. Meth. 175:11-22 (1994); F. D. Ledley etal., Cell Biochem. 18A:226 (1994)).

Another device for providing nucleic acid molecules to a target cell isthe “gene gun”, a ballistic device that projects micro-particles coatedwith nucleic acid molecules directly into the nucleus of cells in vivo.Once within the nucleus, the nucleic acid molecules dissolve from thegold or tungsten microparticle and can be expressed by the target cell.This method has been used effectively to transfer genes directly intothe skin, liver and muscle (N. S. Yang et al., Proc. Natl. Acad. Sci.87:9568-9572 (1990); L. Cheng et al., Proc. Natl. Acad. Sci. USA.90:4455-4459 (1993); R. S. Williams et al., Proc. Natl. Acad. Sci.88:2726-2730 (1991)). Thus, for example, a “gene gun” can be used tointroduce a vector comprising a nucleic acid molecule encoding anosteoprotegerin into heart muscle tissue in culture, and the treatedtissue can be grafted onto, or close to, the damaged portion of ananimal heart to promote formation of new blood vessels.

Osteoprotegerin proteins, or fragments thereof, may be immobilized onto(or within) a surface of an implantable medical device. The modifiedsurface will typically be in contact with living tissue afterimplantation into an animal body. Such implantable medical devices canbe made from, for example, nitrocellulose, diazocellulose, glass,polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran,Sepharose, agar, starch, and nylon. Linkage of the protein to a devicecan be accomplished by any technique that does not destroy thebiological activity of the linked protein, for example by attaching oneor both ends of the protein (i.e., the N-terminal of the protein, and/orthe C-terminal of the protein) to the device. Attachment may also bemade at one or more internal sites in the protein. Multiple attachments(both internal and at the ends of the protein) may also be used. Asurface of an implantable medical device can be modified to includefunctional groups (e.g., carboxyl, amide, amino, ether, hydroxyl, cyano,nitrido, sulfanamido, acetylinic, epoxide, silanic, anhydric,succinimic, azido) for protein immobilization thereto. Couplingchemistries include, but are not limited to, the formation of esters,ethers, amides, azido and sulfanamido derivatives, cyanate and otherlinkages to the functional groups available on osteoprotegerin proteinsor fragments. Osteoprotegerin protein, or fragments thereof, can also beattached non-covalently by the addition of an affinity tag sequence tothe protein, such as GST (see, Smith, D. B., and Johnson, K. S., Gene67:31 (1988)), polyhistidines (see, Hochuli, E., et al., J. Chromatog.411:77 (1987)), or biotin. Such affinity tags may be used for thereversible attachment of the protein to a device. The medical devices ofthe invention described herein can be used, for example, to deliverosteoprotegerin proteins, or fragments thereof, or nucleic acidmolecules encoding osteoprotegerin protein, or fragments thereof, to ananimal body.

With respect to the amount of an osteoprotegerin protein (in a solutionof osteoprotegerin protein) sufficient to promote endothelialmorphogenesis, typically the treatment of a whole animal (e.g., byintravenous injection of an osteoprotegerin protein), or a localized,target population of cells (e.g., by injection of an osteoprotegerinprotein at, or close to, a target tissue or organ within an animal body)utilizes an osteoprotegerin solution having an osteoprotegerinconcentration in the range of from 0.5 to 5 μg/ml. The dosage regime maybe determined empirically without undue experimentation. With respect tothe use of an implantable medical device to deliver an effective amountof an osteoprotegerin protein, typically one or more surfaces of theimplantable medical device is coated with an osteoprotegerin solutionhaving an osteoprotegerin concentration in the range of from 10 to 100μg/ml.

In another aspect, the present invention provides implantable medicaldevices that each include: (a) a device body; and (b) a layer attachedto a surface of the device body, the layer including a molecule(typically a multiplicity of molecules) selected from the groupconsisting of osteoprotegerin and a nucleic acid molecule encodingosteoprotegerin. Osteoprotegerins useful in the methods of the invention(described supra), and nucleic acid molecules encoding osteoprotegerinsuseful in the methods of the invention (described supra), are alsouseful in the devices of the invention.

The implantable medical devices of the invention are adapted to beimplanted into the body of an animal, such as a mammal, including ahuman, during the normal operation of the device. The implantablemedical devices of the invention may be completely implanted into thebody of an animal body (i.e., the entire device is implanted within thebody), or the implantable medical devices may be partially implantedinto an animal body (i.e., only part of the device is implanted withinan animal body, the remainder of the device being located outside of theanimal body). Representative examples of completely implantable medicaldevices include, but are not limited to: cardiovascular devices (such asvascular grafts and stents), artificial blood vessels, artificial bonejoints, such as hip joints, and scaffolds that support tissue growth (insuch anatomical structures as nerves, pancreas, eye and muscle).Representative examples of partially implantable medical devicesinclude: biosensors (such as those used to monitor the level of drugswithin a living body, or the level of blood glucose in a diabeticpatient); percutaneous devices (such as catheters) that penetrate theskin and link a living body to a medical device, such as a kidneydialysis machine; and artificial skin that is applied to a damaged areaof the skin of an animal body (and thereby penetrates the surface of thebody at least by a minimal amount).

FIG. 1 shows a representative medical device 10 of the presentinvention, in the form of an implantable drug delivery device, whichincludes a device body 12 to which is attached a surface layer 14. Inthe embodiment shown in FIG. 1, surface layer 14 has been partiallyremoved to show device body 12 beneath. Device body 12 is indicated byhatching. As shown in the cross-sectional view of medical device 10 inFIG. 2, surface layer 14 includes a surface layer body 16 that definesan internal surface 18, attached to device body 12, and an externalsurface 20. Surface layer 14 may completely or partially cover body 12.

In the representative embodiment of device 10 shown in FIGS. 1 and 2,surface layer 14 is made from a porous matrix 22. FIG. 3 shows arepresentation of porous matrix 22 within which are disposed molecules24 of osteoprotegerin protein (other molecules, such as drugs, ornucleic acid molecules encoding osteoprotegerin, may also be disposedwithin porous matrix 22). Thus, in operation, device 10 is implantedinto a tissue of an animal body where osteoprotegerin molecules 24 arereleased over time and promote endothelial morphogenesis.

The following description of the elements of the implantable medicaldevices of the invention is made with reference to representative device10 shown in FIGS. 1 and 2, but it will be understood that the followingdescription is applicable to the elements of any implantable medicaldevice of the invention. Device body 12 can be made from any suitablematerial. Representative examples of synthetic polymers useful formaking device body 12 include: (poly)urethane, (poly)carbonate,(poly)ethylene, (poly)propylene, (poly)lactic acid, (poly)galactic acid,(poly)acrylamide, (poly)methyl methacrylate and (poly)styrene. Usefulnatural polymers include collagen, hyaluronic acid and elastin.

Surface layer 14 can cover the whole of device body 12, or one or moreparts of device body 12, such as areas of device body 12 where it isdesired to promote endothelial morphogenesis. Surface layer 14 can bemade, for example, from any suitable material that: (a) permitsdeposition therein, or attachment thereto, of osteoprotegerin and/or anucleic acid molecule that encodes an osteoprotegerin; and (b) can beattached to device body 12 (before or after deposition within, orattachment to, surface layer 14 of osteoprotegerin and/or a nucleic acidmolecule that encodes an osteoprotegerin).

Representative examples of materials useful for making surface layer 14include porous matrices. Representative porous matrices useful formaking surface layer 14 include those prepared from tendon or dermalcollagen, as may be obtained from a variety of commercial sources,(e.g., Sigma and Collagen Corporation), or collagen matrices prepared asdescribed in U.S. Pat. Nos. 4,394,370 and 4,975,527. One usefulcollagenous material is termed UltraFiber™, and is obtainable fromNorian Corp. (Mountain View, Calif.).

Certain polymeric matrices may also be employed if desired, theseinclude acrylic ester polymers and lactic acid polymers, as disclosed,for example, in U.S. Pat. Nos. 4,526,909, and 4,563,489. Particularexamples of useful polymers are those of orthoesters, anhydrides,propylene-cofumarates, or a polymer of one or more α-hydroxy carboxylicacid monomers, (e.g., α-hydroxy acetic acid (glycolic acid) and/orα-hydroxy propionic acid (lactic acid)).

By way of representative example, osteoprotegerin, and/or fragmentsthereof that have the ability to promote endothelial morphogenesis, canbe covalently attached to surface layer 14 by any of the following pairsof reactive groups (one member of the pair being present on surfacelayer 14, and the other member of the pair being present on theosteoprotegerin protein(s)): hydroxyl/carboxylic acid to yield an esterlinkage; hydroxyl/anhydride to yield an ester linkage;hydroxyl/isocyanate to yield a urethane linkage.

If surface layer 14 does not possess useful reactive groups, thensurface layer 14 can be treated, for example, with radio-frequencydischarge plasma (RFGD) etching to generate reactive groups in order toallow attachment of osteoprotegerin, or fragments thereof that have theability to promote endothelial morphogenesis, (e.g., treatment withoxygen plasma to introduce oxygen-containing groups; treatment withpropyl amino plasma to introduce amine groups). When an RFGD glowdischarge plasma is created using an organic vapor, deposition of apolymeric overlayer occurs on the exposed surface. RFGD plasma depositedfilms offer several advantages: they are smooth, conformal, and uniform;film thickness is easily controlled and ultrathin films (10-1000Angstroms) are readily achieved, allowing for surface modification of amaterial without alteration to its bulk properties. Moreover, plasmafilms are highly-crosslinked and pin-hole free, and therefore chemicallystable and mechanically durable. RFGD plasma deposition of organic thinfilms has been used in microelectronic fabrication, adhesion promotion,corrosion protection, permeation control, as well as biomaterials. (see,e.g., Ratner, U.S. Pat. No. 6,131,580).

Typically, nucleic acid molecules that encode osteoprotegerin, or encodean osteoprotegerin fragment that has the ability to promote endothelialmorphogenesis, are non-covalently attached to surface layer 14, e.g.,nucleic acid molecules can be disposed within the pores of a porousmaterial that is used to form surface layer 14. The nucleic acidmolecules diffuse out of porous surface layer 14, when device 10 isimplanted into an animal body, and are taken up by adjacent cellswherein the osteoprotegerin encoded by the nucleic acid molecules isexpressed.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

EXAMPLE 1

This Example shows that an osteoprotegerin-IgGFc protein fusion is aseffective as fibroblast growth factor at inducing the formation of bloodvessels in an in vivo impregnated sponge assay.

C57BL6 mice were implanted with polyvinyl alcohol sponges presoaked inone of the following solutions which each had a concentration of 100ng/ml: a solution of bovine fibroblast growth factor (bFGF); a solutionof the Fc portion of human IgG (FC); and a solution of a hybrid proteincomposed of the Fc portion of human IgG fused to osteoprotegerin(OPG-FC). The control was phosphate buffered saline (PBS).

After the sponges had been implanted subcutaneously in the backs of themice for a period of fourteen days, the sponges were removed and thenumber of blood vessels growing into the sponges were counted under amicroscope. The results are shown in FIG. 4, and show thatosteoprotegerin-IgGFc protein fusion was as effective as fibroblastgrowth factor at inducing the formation of blood vessels.

EXAMPLE 2

This Example shows that osteoprotegerin protein induces the formation ofblood vessels in an in vitro rat aortic ring assay.

Thoracic aorta was excised from five to ten week old Fischer 344 malerats. The periaortic fibroadipose tissue was dissected and the cleanedaorta was cross-sectioned to yield rings of 1-2 mm in length. The ringswere embedded in collagen gels. The collagen gels were prepared bymixing eight volumes of 1 mg/ml collagen with one volume of 10× MinimalEssential Medium (MEM, Invitrogen), pH 4.0, and one volume of 23.4 mg/mlNaHCO₃. The embedded aortic rings were transferred to 16 mm wells thateach contained 0.5 ml of serum-free endothelial basal medium (sold byInvitrogen as MCDB 131). The medium was changed three times per weekstarting at day three. The cultures were treated with a 140 nM solutionof human recombinant osteoprotegerin. The control was aortic sectionstreated with Phosphate Buffered Saline only. The number of blood vesselsgrowing from the aortic rings into the collagen gel were counted on days3, 6 and 8 after embedding into the collagen gel.

As shown in FIG. 5, the number of blood vessels growing into thecollagen gel was significantly greater in the presence ofosteoprotegerin compared to the control culture lacking osteoprotegerinin the gel.

EXAMPLE 3

This example describes a representative hybridization protocol that canbe used to identify nucleic acid molecules that encode anosteoprotegerin, or a portion of an osteoprotegerin that has the abilityto promote endothelial morphogenesis, and that hybridize to thecomplement of the nucleic acid molecule consisting of the nucleic acidsequence set forth in SEQ ID NO:1, under defined hybridizationconditions. In this Example, the complement of the nucleic acid moleculeconsisting of the nucleic acid sequence set forth in SEQ ID NO:1 is usedas probe.

Hybridization solution should preferably be prepared and filteredthrough a 0.45-micron disposable cellulose acetate filter. Thecomposition of the hybridization solution is 6×SSC, 5× Denhardt'sreagent, 0.5% sodium dodecyl sulfate (SDS), 100 μg/ml denatured,fragmented salmon sperm DNA. The abbreviation “SSC” refers to a bufferused in nucleic acid hybridization solutions. One liter of the 20×(twenty times concentrate) stock SSC buffer solution (pH 7.0) contains175.3 g sodium chloride and 88.2 g sodium citrate. When ³²P-labeled cDNAor RNA is used as a probe, poly(A)⁺ RNA at a concentration of 1 μg/mlmay be included in the hybridization solution to prevent the probe frombinding to T-rich sequences that are found fairly commonly in eukaryoticDNA.

The nitrocellulose filter or nylon membrane containing the target DNA isfloated on the surface of a tray of 6×SSC until it becomes thoroughlywetted from beneath. The filter is submerged for about 2 minutes. Thewet filter is placed into a heat-sealable bag, and 0.2 ml ofhybridization solution is added for each square centimeter ofnitrocellulose filter or nylon membrane.

As much air as possible is squeezed from the bag, and the open end ofthe bag is sealed with a heat sealer. The bag is incubated for 1-2 hourssubmerged at the desired temperature (typically no higher than thehybridization temperature). It is desirable to agitate the bag.

If the radiolabeled probe is double-stranded, then it should preferablybe denatured by heating for 5 minutes at 100° C. Single-stranded probeneed not be denatured. The denatured probe is chilled rapidly in icewater. Ideally, probe having a specific activity of 10⁹ cpm/μg, orgreater, is used. Hybridization is carried out for the desired timeperiod (e.g., for a period of from 12 hours to 24 hours) at 50° C.,typically using 1-2 μg/ml radiolabeled probe.

The bag containing the filter is removed from the water bath, and thebag is opened by cutting off one corner with scissors. The denaturedprobe is added to the hybridization solution, and then as much air aspossible is squeezed from the bag. The bag is resealed with the heatsealer so that as few bubbles as possible are trapped in the bag. Toavoid radioactive contamination of the water bath, the resealed bagshould be sealed inside a second, noncontaminated bag.

The bag is submerged in a water bath and incubated for the requiredperiod of hybridization. The bag is removed from the water bath and onecorner is cut off. The hybridization solution is poured into a containersuitable for disposal, and then the bag is cut along the length of threesides. The filter is removed and immediately submerged in a traycontaining several hundred milliliters of 2×SSC and 0.5% SDS at roomtemperature (no higher than 25° C.). The filter should not be allowed todry out at any stage during the washing procedure.

After 5 minutes, the filter is transferred to a fresh tray containingseveral hundred milliliters of 2×SSC and 0.1% SDS and incubated for 15minutes at room temperature (no higher than 25° C.) with occasionalgentle agitation. The filter is then washed at the desired stringency,i.e., in the desired concentration of SSC and at the desired temperaturefor the desired period of time. If, for example, nucleic acid moleculesthat hybridize to the probe at a temperature of 60° C. in 5×SSC for 30minutes are sought, then the filter is washed in 5×SSC at 60° C. for 30minutes, i.e., the radiolabelled probe is washed off nucleic acidmolecules (immobilized on the filter) that do not hybridize to the probeunder conditions of 5×SSC at 60° C. for 30 minutes. It is understoodthat the stringency of the hybridization step is not higher than thestringency of the wash step.

After washing, most of the liquid is removed from the filter by placingit on a pad of paper towels. The damp filter is placed on a sheet ofSaran Wrap, and adhesive dot labels marked with radioactive ink areapplied at several asymmetric locations on the Saran Wrap. These markersserve to align the autoradiograph with the filter. The labels should becovered with Scotch Tape which prevents contamination of the film holderor intensifying screen with the radioactive ink. Radioactive ink is madeby mixing a small amount of ³²P with waterproof black drawing ink.

The filter is covered with a second sheet of Saran Wrap, and the filteris exposed to X-ray film (Kodak XAR-2 or equivalent) to obtain anautoradiographic image. The exposure time should be determinedempirically.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method for promoting endothelial morphogenesis in vitro, saidmethod comprising the step of providing to endothelial cells cultured ona porous matrix or scaffold that supports tissue growth in vitro anamount of an osteoprotegerin sufficient to promote endothelialmorphogenesis.
 2. The method of claim 1, wherein the osteoprotegerin isat least 70% identical to the osteoprotegerin consisting of the aminoacid sequence set forth in SEQ ID NO:
 2. 3. The method of claim 1,wherein the osteoprotegerin is at least 80% identical to theosteoprotegerin consisting of the amino acid sequence set forth in SEQID NO:
 2. 4. The method of claim 1, wherein the osteoprotegerin is atleast 90% identical to the osteoprotegerin consisting of the amino acidsequence set forth in SEQ ID NO:
 2. 5. The method of claim 1, whereinthe endothelial cells are cultured in a liquid medium that comprises theosteoprotegerin.
 6. The method of claim 1, wherein the endothelialmorphogenesis is formation of capillaries.
 7. The method of claim 1,wherein the endothelial morphogenesis is formation of an endotheliallining in a blood vessel.
 8. The method of claim 7, wherein theendothelial lining is formed in an artificial blood vessel.
 9. Themethod of claim 7, wherein the endothelial lining is formed in a naturalblood vessel.